Effects of ions and ionic channel activators or blockers on release of α-MSH from perifused rat hypothalamic slices

Abstract

The involvement of sodium and chloride ions in the process of α-melanocyte-stimulating hormone (α-MSH) release from hypothalamic neurons was investigated using perifused rat hypothalamic slices. Three different stimuli were found to increase α-MSH release from hypothalamic slices: high K + concentration (50 mM), veratridine (50 μM), and the Na + K +- ATPase inhibitor ouabain (1 mM). Spontaneous or K +-evoked α-MSH release was insensitive to the specific Na + channel blocker tetrodotoxin (TTX; 1.5 μM) and to the blocker of K + channels tetraethylammonium (TEA; 30 mM) or 4-aminopyridine (4-AP; 4 mM). In contrast, blockage of ouabain-sensitive Na + K +- ATPase increased the resting level of α-MSH and caused a dramatic potentiation of K +-evoked α-MSH release. The Na + channel activator veratridine (50 μM) triggered α-MSH release. This stimulatory effect was blocked by TTX and prolonged by TEA application, indicating the occurrence of voltage-sensitive Na + and K + channels on α-MSH neurons. Replacement of Na + by impermeant choline ions from 95 to 60 mM did not alter K +-evoked α-MSH release. Conversely, dramatic reduction of the external Na + concentration to 16 mM caused a robust increase of α-MSH secretion from hypothalamic neurons, likely through activation of the Na + Ca 2+ exchange system. These data indicate that the depolarizing effect of K + results from direct activation of voltage-operated Ca 2+ channels. The lack of effect of TEA on basal α-MSH release prompted us to investigate the possible involvement of chloride ions in the regulation of the spontaneous activity of α-MSH neurons. Substitution of Cl − for impermeant acetate ions did not affect basal or K +-evoked α-MSH release. In contrast, administration of TEA in low Cl − medium stimulated α-MSH secretion. The response of hypothalamic neurons to TEA in low Cl − medium was totally suppressed by TTX, indicating a Na +-dependent regenerative spontaneous activity. The GABA A agonist, muscimol (100 μM), caused a marked inhibition of the stimulated α-MSH release induced by concomitant administration of veratridine and TEA. The effect of muscimol was reversed by bicuculline (100 μM), a specific GABA A antagonist. Taken together, these data indicate that high K +, veratridine and ouabain trigger α-MSH release through distinct mechanisms: veratridine activates Na + conductance; K + depolarization directly recruits voltage-operated Ca 2+ channels which do not belong to the L-type; ouabain likely increases the internal Na + concentration. Our data also suggest the occurrence of an inhibitory mechanism, dependent on Cl − ions, likely mediated through activation of the GABA A receptor, which may play a pivotal role in the regulation of α-MSH release by hypothalamic neurons.